Even increased the with contemporary anti-retroviral therapy (ART) regimens that the survival rate , HIV remain s an enormous health burden disease by eliminating proviral DNA, which is present in the genome of infected host cells. suppress viral . Unfortunately, ART has replication and have failed to cure The persistence of the viral genome in tissues with the potential to become reactivated, when ART is discontinued, underscores the importance of alternative, perhaps companion, strategies for eradication of the HIV genome from the latently infected reservoirs. The possibility of a curative treatment for HIV infection has been energized by the ?Berlin patient? and more recently the ?London patient?, who both received stem cell transplants from a CCR5?32-homozygous donor after radiation and chemotherapy for leukemia and have been undetectable for the virus in his blood for 12 and 2 years since stopping ART after the transplants. CCR5 is a cellular protein that serves a co-receptor for HIV infection, therefore its genetic inactivation prevents viral infection and its spread throughout the body of the infected individual. In previous studies, we successfully applied CRISPR- Cas9 gene editing strategy to target and eradicate integrated HIV sequences in in vitro cell culture models, ex vivo cultured HIV+ patient derived and in vivo in HIV-infected humanized mice model. We presented data at CROI 2019 and here of in vivo editing of the SIV genome in blood of SIV-infected macaques after i.v. injection of AAV9/CRISPR-Cas9. In this multi Principal investigator (mPi) grant application, we will test the hypothesis that employment of a combined gene editing platform can effectively excise SIV proviral DNA and further editing of the CCR5 gene will remove viral targets in the animal leading to a reduction in the functional viral reservoir and/or complete elimination of replication competent virus in the treated animals. To this end, we will employ CRISPR-Cas9 technology for sequential targeting and permanent inactivation of both SIV and CCR5 in an SIV-infected non-human primate model. To achieve our goals, we investigators have formed a team of experienced with extensive experience in in vivo SIV animal models and immunology (T. Burdo, mPi, Temple University), gene editing technologies for eradicating virus from host genomes in in vitro cell cultures and in vivo animal models novel assays In this application, we will establish the CCR5 and dual SIV and CCR5 gene editing platforms in SIV-infected non-human primates and determine the immunological and virological effects and the underlying mechanism of cure or delayed viral rebound after gene editing and antiretroviral therapy interruption (ATI). We will use single and then a sequential dual editing targeting the cellular gene (CCR5) followed by the SIV proviral DNA. The outcome of these studies will offer valuable insight into the potential of CRISPR-Cas9 gene editing for consideration in the HIV cure strategies. (K. Khalili, mPi, Temple University), and (J. Karn, co-I, Case Western Reserve University). measuring the inducible viral RNA reservoir using